ANGIOTENSIN II-MEDIATED EXPRESSION OF P27(KIP1) INDUCTION OF CELLULARHYPERTROPHY IN RENAL TUBULAR CELLS DEPEND ON THE GENERATION OF OXYGENRADICALS

Background. Angiotensin II (Ang II) induces hypertrophy of cultured pr oximal tubular cells. We have previously demonstrated that this Ang II -mediated hypertrophy occurs in the G(1)-phase of the cell cycle and d epends on the induction of p27(Kip1),an inhibitor of G(1)-phase cyclin /cyclin-dependent kinase complexes. The present study was undertaken t o investigate whether Ang II may stimulate superoxide anions (O-2(.)) formation in cultured LLC-PK1 and cultured mouse proximal tubule (MCT) cells, and to gain further insight into a potential relationship betw een O-2(.) and cell cycle regulation. Methods. Reactive oxygen species were measured with the lucigenin method in intact cells. The effects of various inhibitors were tested on Ang II-induced O-2(.) production. Cells were transiently transfected with phosphorothioate-modified rat p22phox antisense oligonucleotides to investigate the potential role of NAD(P)H oxidase. Expression of p22phox mRNA after Ang II-treatment was detected with Northern blots. Incorporation of [H-3]leucine into d e novo synthesized proteins was used as a parameter of cell hypertroph y. Expression of p27(Kip1) was evaluated in cell lysates by Western bl otting. Results. Ang II stimulated the accumulation of O-2(.) in tubul ar cells; however, an addition of two different antioxidants completel y abolished measurable O-2(.). This effect was transduced by angiotens in receptor type-1 (AT(1)) and was inhibited by a flavoprotein inhibit or (DIP) or p22phox antisense oligonucleotides, indicating the involve ment of membrane NAD(P)H oxidase. Ang II-stimulated de novo protein sy nthesis was attenuated by DIP, antioxidants, and p22phox antisense oli gonucleotides. The Ang II-induced expression of p27(Kip1) protein and cellular hypertrophy were reduced by similar treatments. Generation of O-2(.) by xanthine supplementation also stimulated p27(Kip1) expressi on and induced hypertrophy in LLC-PK1 cells. Conclusion. This study pr ovides the first evidence, to our knowledge, that Ang II induces O-2. in cultured tubular cells. Ang II-mediated activation of membrane boun d NAD(P)H oxidase, probably by an increase in p22phox transcripts, is likely responsible for this induction. Generation of O-2(.) subsequent ly induces p27(Kip1) expression and stimulates hypertrophy, suggesting a novel mechanism of how Ang II can modulate cell cycle regulation.